Curation Information

The Escherichia coli RutR transcription factor binds at targets within genes as well as intergenic regions.;Shimada T, Ishihama A, Busby SJ, Grainger DC;Nucleic acids research 2008 Jul; 36(12):3950-5 [18515344]
RutR [P0ACU2, view regulon]
Reported TF sp.
Escherichia coli BW25113
Reported site sp.
Escherichia coli BW25113
Created by
Erill Lab
Curation notes

Experimental Process

ChIP-chip to detect sites. Motif discovery and site search to identify sites. EMSA to validate targets. Northern blot to validate regulation.

ChIP assay conditions
For experiments with exponentially growing cells, overnight cultures of E. coli strain BW25113 or JW0998 were diluted 1:100 into fresh medium either with or without uracil, and grown for ∼4 hours to an OD650 of 0.3–0.4.
ChIP notes
Assays were done either in triplicate (−uracil) or in duplicate (+uracil). Briefly, cultures of E. coli BW25113 and, as a control, JW0998 ΔrutR were grown to mid-log phase at 37°C. Cells were then treated with 1% formaldehyde and broken open by sonication which also fragments cross-linked nucleoprotein. Cross-linked RutR–DNA complexes were immunoprecipiated from cleared lysates of BW25113 using anti-RutR rabbit polyclonal anti-serum, and parallel samples were isolated from control JW0998 ΔrutR cells. Cross-links were then reversed and immunoprecipitated DNA was purified. DNA samples isolated from BW25113 cells and the control ΔrutR cells were labelled with Cy5 and Cy3, respectively. To identify segments of DNA specifically associated with RutR, the two labelled samples were combined and hybridised to a 22 000 feature DNA microarray (Oxford Gene Technology, Oxford, UK). For each probe, the Cy5/Cy3 ratio was measured and this was plotted against the corresponding position on the E. coli BW25113 chromosome, creating a profile of RutR binding (Figure 1). We then selected ‘peaks’, formed by two or more consecutive probes, with a Cy5/Cy3 ratio of >2.5. To increase the stringency of our search, we discarded the small number of peaks where the RutR-binding signal was not reduced at least two-fold when uracil was added to cultures. The centre of each peak is defined as the centre of the probe within the peak that had the highest Cy5/Cy3 signal.

Transcription Factor Binding Sites


Gene Regulation

Regulated genes for each binding site are displayed below. Gene regulation diagrams show binding sites, positively-regulated genes, negatively-regulated genes, both positively and negatively regulated genes, genes with unspecified type of regulation. For each indvidual site, experimental techniques used to determine the site are also given.

Site sequence Regulated genes Gene diagram Experimental techniques TF function TF type
... ... ves cho spy
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details EMSA (ECO:0001807) - Experimental technique details Northern blot (ECO:0005653) - repressor dimer